The long term goal of this grant is to advance understanding of chemotaxis, the ability of cells to sense chemical gradients and move directionally. Studies oi Dictyostelium discoideum, with its accessible biochemistry, cell biology, and genetics, have contributed enormously to the current understanding of chemotaxis. Briefly, in Dictyostelium and human neutrophils, chemotaxis is mediated by GPCRs linked to specific heterotrimeric G-proteins, which trigger downstream signaling events to occur selectively on the side of the cell facing the higher concentration of chemoattractant. For example, the accumulation of phosphatidylinositol 3,4, 5-tris phosphate (PIP3) is sharply localized to the leading edge of the cell. Recent studies have shown that PIP3 acts in parallel with other signaling events in a network that biases the motility machinery of the cell. In the Progress Report, we describe our studies of tsunami which plays a key role in cell polarity. We define me central roles of pianissimo (piaA), a subunit of Tor Complex 2 (TorC2), and a phospholipase A2, plaA. We report single molecule imaging studies of receptors and G-proteins revealing the earliest steps in gradient sensing. We describe the results of a large forward genetic screen that uncovered over twenty novel chemotaxis genes. We also report on a genomic library complementation approach to identify chemically-induced mutants. The latter two projects, involving considerable risk and long term commitment, were made possible by the MERIT award. The plans for the second half of the grant are presented. We will continue to unravel the network of signaling pathways that mediate chemotaxis, exploiting the ability to combine gene disruptions in Dictyostelium, along with careful biochemical and cell biological experiments. We will ask whether key elements of the pathways are conserved in neutrophils. To define the roles of the new chemotaxis genes we have identified, we will assess a series of well-characterized physiological responses associated with chemotaxis such as PIP3 production, actin polymerization, and phosphorylation of PKB substrates and carry out and analyze time-lapse images of the cells undergoing directed migration. We will pursue the single molecule imaging studies to achieve and refine direct imaging individual G-protein activation events, hopefully creating as powerful an experimental tool as single channel recording of ion channel openings. RELEVANCE (See instructions): Chemotaxis mediates many physiological processes including embryogenesis, immune response, wound healing, and stem cell homing and plays a key role in the pathology of inflammatory diseases and cancer metastasis. A detailed understanding of chemotaxis, which involves links between signal transduction and cell motility networks, would provide an incredibly powerful tool for designing rational interventions for improving health and defeating disease.